**1. Introduction**

In the last few years, petroleum-based food packaging materials have been replaced with biodegradable biopolymers as a result of circular economy and sustainability [1–5]. Thus, the utilization of protein and polysaccharide-based biopolymer hybrid nanostructure materials in the food industry has been increased due to their non-toxicity, biodegradability, ability to form gels, encapsulate and deliver bioactive compounds such as essential oils [6–8]. Such novel biopolymer-based food packaging systems are suitable for active food packaging applications. Active food packaging is defined as "packaging in which subsidiary constituents have been deliberately included in or on either the packaging material or the package headspace to enhance the performance of the package system" [4].

Alginate (ALG) has become one of the most popular natural polysaccharides extensively used in the development of delivery systems for food bioactive ingredients. The suitability of this material for such purposes is due to its ionic crosslinking ability, pH responsiveness, excellent biocompatibility, biodegradability, and low price [5]. Alginate (ALG) is an unbranched anionic polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked by glycosidic bonds. The structure of alginate depends

**Citation:** Giannakas, A.E.; Salmas, C.E.; Moschovas, D.; Zaharioudakis, K.; Georgopoulos, S.; Asimakopoulos, G.; Aktypis, A.; Proestos, C.; Karakassides, A.; Avgeropoulos, A.; et al. The Increase of Soft Cheese Shelf-Life Packaged with Edible Films Based on Novel Hybrid Nanostructures. *Gels* **2022**, *8*, 539. https://doi.org/10.3390/ gels8090539

Academic Editor: Bjørn Torger Stokke

Received: 30 July 2022 Accepted: 23 August 2022 Published: 26 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

primarily on the monomer composition, the M/G ratio, the polymer sequence, and the molecular weight of the linear chain. The structure of alginate and the M/G ratio are crucial for its capability to deliver bioactive compounds. For example, a higher concentration of G blocks generates more rigid hydrogels with larger pores [6], which leads to the easier release of immobilized bioactive components from the polymer matrix. On the contrary, higher M block content is more suitable for the formulation of softer edible films and coatings with lower gas permeability [7,8].

Under the same spirit, there is a trend to replace the "commonly" used antioxidant and/or antimicrobial chemical agents such as Butylated hydroxytoluene (BHT) and Butylated Hydroxyanisole (BHA) which are added directly to the food. The use of such chemicals was replaced with the use of essential oils [9,10] or other bioactive phytochemicals [11] in active packaging film gels, and coating is taking place. Essential oil loss due to evaporation phenomena was reduced using various nanomaterials as nanocarriers in food delivery systems and active food packaging applications. Such materials were developed based on the food nanotechnology concept [12–14]. Nanoclays, such as montmorillonite [15–17] and halloysite [18–20] were used both as reinforcements and as essential oil nanocarriers for controlled released applications [21]. These nanoclay based essential oil nanocarriers were incorporated into polymer [21–23] or biopolymer [16] networks. The new composite materials were promising for active packaging film applications with antioxidant and/or antimicrobial activity. Natural zeolite (NZ) is another, abundant nanomaterial promising for food preservation and food packaging applications [24] as is reported in the literature [25–27]. Rešˇcek et al., 2018 [28] developed double-layered polyethylene/caprolactone packaging films modified with zeolite and magnetite. It was shown that the addition of zeolite improved the mechanical and barrier properties of obtained films. Youssef et al., 2019 [29] prepared carboxymethyl cellulose/polyvinyl alcohol films modified with zeolite which was firstly doped with Ag and Au ions. It was shown that the addition of this modified zeolite enhanced the mechanical, barrier and antimicrobial properties of the obtained packaging films. Recently, Nascimento Souza et al., 2020 [30] prepared chitosan packaging film and used zeolite as an ethylene scavenger. To the best of our knowledge, there is no study on the use of natural zeolite (NZ) as nanoreinforcement and/or essential oil nanocarrier in ALG based film preparation.

Cottage cheese is a highly consumed type of cheese which, however, is easily acidified. Because of its high moisture content i.e., about 75%, and pH values over 4.5, the shelf-life of this product is restricted to 15 days [31]. It is well documented that various types of spoilage bacteria, yeasts, and molds that may develop on the cheese surface during storage can influence the shelf life of cheese, particularly in the case of soft and spread cheese. To prevent damage and spoilage, soft and fresh cheese are currently packaged with active cheese technology [32].

In this study natural zeolite (NZ) was firstly modified with thyme essential oil (TO) and produced a novel TO@NZ hybrid nanostructure. These nanostructures were characterized with XRD analysis and FTIR spectrometry. They were directly added to sodium alginate (ALG) plasticized with glycerol (G) hydrogels and produced novel ALG/G/TO@NZ active packaging films. The TO@NZ hybrid nanostructure content was fixed to 5, 10, and 15% wt. The properties of these films were compared with the properties of films prepared with pure NZ. The obtained ALG/G/NZ and ALG/G/TO@NZ films were also characterized with XRD analysis and FTIR spectrometry. Moreover, they were tested for their mechanical and water/oxygen barrier properties. The antioxidant and antimicrobial capacity of the obtained films was also evaluated. Finally, the most active films were used as packaging films to extend the shelf-life of soft cheese. The innovation of the current study can be summarized in the following three points: (1) modification/preparation of a rich in thymol content NZ nanostructure via a green evaporation/adsorption method, (2) development of edible active packaging films based on an ALG/G biopolymer matrix by using the novel TO@NZ nanostructure, and (3) use of such edible active packaging films to extend the self–life of a soft cottage-cheese.

#### **2. Results and Discussion** *2.1. GC-MS Results*

films to extend the self–life of a soft cottage-cheese.

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used as packaging films to extend the shelf-life of soft cheese. The innovation of the current study can be summarized in the following three points: (1) modification/preparation of a rich in thymol content NZ nanostructure via a green evaporation/adsorption method, (2) development of edible active packaging films based on an ALG/G biopolymer matrix by using the novel TO@NZ nanostructure, and (3) use of such edible active packaging

#### *2.1. GC-MS Results* The results of the GC-MS analysis of the TO as received and of the remaining after

**2. Results and Discussion** 

*2.2. DSC Results* 

The results of the GC-MS analysis of the TO as received and of the remaining after the first stage distillation process TO are summarized in Tables S1 and S2. The main substances of the TO as received are p-cymene 12.3%, D-limonene 16.5%, and thymol 56.7% (see Table S1). At the end of the first stage distillation process, the remaining TO does not contain p-cymene and D-limonene and contains 86.7% thymol (see Table S2). the first stage distillation process TO are summarized in Tables S1 and S2. The main substances of the TO as received are p-cymene 12.3%, D-limonene 16.5%, and thymol 56.7% (see Table S1). At the end of the first stage distillation process, the remaining TO does not contain p-cymene and D-limonene and contains 86.7% thymol (see Table S2).

#### *2.2. DSC Results* Figure 1 presents DSC plots of pure NZ, TO@NZ, and TO\_NZ hybrid nanostructures

Figure 1 presents DSC plots of pure NZ, TO@NZ, and TO\_NZ hybrid nanostructures in the range of 50–250 ◦C. in the range of 50–250 °C.

**Figure 1.** DSC plots of (1) pure NZ, (2) TO\_NZ hybrid nanostructure and (3) TO@NZ hybrid

**Figure 1.** DSC plots of (1) pure NZ, (2) TO\_NZ hybrid nanostructure and (3) TO@NZ hybrid nanostructure.

nanostructure. All curves exhibit a small, wide, and broad peak starting at around above 100 °C. This peak is attributed to the exothermic water evaporation process. In the case of TO\_NZ and TO@NZ hybrid nanostructures two sharp exothermic peaks at approximately 185 °C and 230 °C are observed correspondingly. Additionally, in the case of TO\_NZ hybrid nanostructure, there is also a wide broad peak starting above 200 °C. Precisely in the case of TO\_NZ hybrid nanostructure the sharp peak at 185 °C does not ends and continues above 200 °C. This peak at 185 °C corresponds to p-Cymene and D-Limonene molecules' evaporation while the peak at 230 °C corresponds to the thymol molecules' evaporation [33–35]. This observation indicates that molecules that existed in the TO\_NZ hybrid nanostructure are of different kinds of molecules that existed in the TO@NZ hybrid nanostructure. In the case of TO\_NZ hybrid nanostructures, Limonene, Cymene, and Thy-All curves exhibit a small, wide, and broad peak starting at around above 100 ◦C. This peak is attributed to the exothermic water evaporation process. In the case of TO\_NZ and TO@NZ hybrid nanostructures two sharp exothermic peaks at approximately 185 ◦C and 230 ◦C are observed correspondingly. Additionally, in the case of TO\_NZ hybrid nanostructure, there is also a wide broad peak starting above 200 ◦C. Precisely in the case of TO\_NZ hybrid nanostructure the sharp peak at 185 ◦C does not ends and continues above 200 ◦C. This peak at 185 ◦C corresponds to p-Cymene and D-Limonene molecules' evaporation while the peak at 230 ◦C corresponds to the thymol molecules' evaporation [33–35]. This observation indicates that molecules that existed in the TO\_NZ hybrid nanostructure are of different kinds of molecules that existed in the TO@NZ hybrid nanostructure. In the case of TO\_NZ hybrid nanostructures, Limonene, Cymene, and Thymol molecules were adsorbed while in the case of TO@NZ hybrid nanostructures the Limonene and Cymene were removed from TO during the distillation process, and thymol was mainly adsorbed. Additionally, in the case of TO\_NZ hybrid nanostructures, higher amounts of Limonene and Cymene molecules were adsorbed.

> The DSC results indicate that the TO evaporation process led to the adsorption of higher quantities of D-Limonene and p-Cymene than thymol on the NZ surface (TO\_NZ hybrid nanostructure). This happens probably because of their lower evaporation temperature. On the contrary, when D-Limonene and p-Cymene molecules were removed from TO via the distillation process, thymol is the main substance which was adsorbed on the NZ surface (TO@NZ hybrid nanostructure). In other words, from DSC plots, it is clear that

the TO\_NZ hybrid nanostructure was rich in D-Limonene and p-Cymene molecules while TO@NZ was rich in thymol molecules. the TO\_NZ hybrid nanostructure was rich in D-Limonene and p-Cymene molecules while TO@NZ was rich in thymol molecules.

mol molecules were adsorbed while in the case of TO@NZ hybrid nanostructures the Limonene and Cymene were removed from TO during the distillation process, and thymol was mainly adsorbed. Additionally, in the case of TO\_NZ hybrid nanostructures, higher

The DSC results indicate that the TO evaporation process led to the adsorption of higher quantities of D-Limonene and p-Cymene than thymol on the NZ surface (TO\_NZ hybrid nanostructure). This happens probably because of their lower evaporation temperature. On the contrary, when D-Limonene and p-Cymene molecules were removed from TO via the distillation process, thymol is the main substance which was adsorbed on the NZ surface (TO@NZ hybrid nanostructure). In other words, from DSC plots, it is clear that

### *2.3. XRD Analysis 2.3. XRD Analysis*

The XRD plots of the as received NZ and of the modified TO@NZ hybrid nanostructure are presented in Figure 2. The XRD plots of the as received NZ and of the modified TO@NZ hybrid nanostructure are presented in Figure 2.

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amounts of Limonene and Cymene molecules were adsorbed.

**Figure 2.** XRD plots of as received NZ and TO@NZ hybrid nanostructure. **Figure 2.** XRD plots of as received NZ and TO@NZ hybrid nanostructure.

The observed reflections in patterns of both NZ and TO@NZ materials are attributed to Heulandite Ca(Si7Al2)O16 × 6H2O monoclinic crystal phase (PDF-41-1357). This means The observed reflections in patterns of both NZ and TO@NZ materials are attributed to Heulandite Ca(Si7Al2)O<sup>16</sup> <sup>×</sup> 6H2O monoclinic crystal phase (PDF-41-1357). This means that the adsorption of TO into NZ did not affect the crystal phase. *Gels* **2022**, *8*, x FOR PEER REVIEW 5 of 24

> that the adsorption of TO into NZ did not affect the crystal phase. The XRD plots of pure ALG/G, ALG/G/xNZ, and ALG/G/xTO@NZ nanocomposite The XRD plots of pure ALG/G, ALG/G/xNZ, and ALG/G/xTO@NZ nanocomposite films are presented in Figure 3 (where x is the nanostructure composition).

**Figure 3.** XRD plots of: (1) ALG/G, (2) ALG/G/5NZ, (3) ALG/G/10NZ, (4) ALG/G/15NZ, (5) ALG/G/5TO@NZ, (6) ALG/G/10TO@NZ AND (7) ALG/G/15TO@NZ obtained films. **Figure 3.** XRD plots of: (1) ALG/G, (2) ALG/G/5NZ, (3) ALG/G/10NZ, (4) ALG/G/15NZ, (5) ALG/G/5TO@NZ, (6) ALG/G/10TO@NZ AND (7) ALG/G/15TO@NZ obtained films.

changes of the ALG crystallinity were observed with the addition of either NZ or TO@NZ hybrid nanostructures. Moreover, after an initial additive loading into the polymeric matrix, as the % wt. content of NZ or TO@NZ hybrid nanostructure increases the reflections of zeolite's crystal phase are increase. This indicates that the higher dispersion of such

materials in the ALG/G film is obtained only for low % wt. loadings i.e., <10% wt.

Unplasticized alginate films exhibited two broad peaks with central positions at 2θ = 13.5° and 21.6° [36,37]. As it is observed in Figure 3, in the case of such ALG/G films the peak at 2θ = 13.5° disappeared indicating a lower proportion of the amorphous structure

Line (1) in Figure 4 represents the FTIR spectra of the as received TO. In the same figure, Line (2) is assigned to the as received natural zeolite FTIR spectra, and Line (3) to

the modified rich in thymol natural zeolite TO@NZ.

*2.4. FTIR Spectroscopy* 

Unplasticized alginate films exhibited two broad peaks with central positions at 2θ = 13.5◦ and 21.6◦ [36,37]. As it is observed in Figure 3, in the case of such ALG/G films the peak at 2θ = 13.5◦ disappeared indicating a lower proportion of the amorphous structure with larger chain distances. This is a result of water and glycerol de-structuration [36]. No changes of the ALG crystallinity were observed with the addition of either NZ or TO@NZ hybrid nanostructures. Moreover, after an initial additive loading into the polymeric matrix, as the % wt. content of NZ or TO@NZ hybrid nanostructure increases the reflections of zeolite's crystal phase are increase. This indicates that the higher dispersion of such materials in the ALG/G film is obtained only for low % wt. loadings i.e., <10% wt.
